WO1994012214A1 - Anticorps humanises reagissant avec la cd-18 - Google Patents

Anticorps humanises reagissant avec la cd-18 Download PDF

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Publication number
WO1994012214A1
WO1994012214A1 PCT/US1993/011611 US9311611W WO9412214A1 WO 1994012214 A1 WO1994012214 A1 WO 1994012214A1 US 9311611 W US9311611 W US 9311611W WO 9412214 A1 WO9412214 A1 WO 9412214A1
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immunoglobulin
humanized
human
antibody
humanized immunoglobulin
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PCT/US1993/011611
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English (en)
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Man Sung Co
Nicholas F. Landolfi
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Protein Design Labs, Inc.
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Priority to AU57326/94A priority Critical patent/AU5732694A/en
Publication of WO1994012214A1 publication Critical patent/WO1994012214A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2839—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily
    • C07K16/2845—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily against integrin beta2-subunit-containing molecules, e.g. CD11, CD18
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates generally to the combination of recombinant DNA and monoclonal antibody technologies for developing novel biologies and, more particularly, for example, to the production of non- immunogenic (in humans) immunoglobulins specific for the CD18 protein and their uses -in vitro and in vivo.
  • adhesion molecules generally glycoproteins, expressed on cell membranes. Often, an adhesion molecule on one cell type will bind to another adhesion molecule expressed on a different cell type, forming a receptor counter-receptor pair.
  • adhesion molecules Three very important classes of adhesion molecules are the integrins, selectins, and immunoglobulin (Ig) superfamily members (see Springer, Nature 346. 425 (1990) ; Osborn, Cell 62. 3 (1990); Hynes, Cell -69, 11 (1992), all of which are incorporated herein by reference) . These molecules are especially vital to the interaction of leukocytes and platelets with themselves and with the extracellular matrix and vascular endothelium.
  • Integrins are heterodimeric transmembrane glycoproteins consisting of an a chain (120-180 kD) and a ⁇ chain (90-110 kD) , generally having short cytoplasmic domains.
  • the ⁇ subunits all share sequence homology and motifs with each other, as do the ⁇ subunits.
  • the three known integrins containing the ⁇ subunit designated ⁇ 2 are important to the function of T cells, neutrophils and monocytes.
  • LFA-l (a L ⁇ 2 ) s widely distributed on lymphocytes, granulocytes and monocytes.
  • ICAM-1 Its counter-receptor is ICAM-1 (and perhaps of lesser importance, ICAM-2) an Ig family molecule which is expressed on many cells including leukocytes and is up-regulated on vascular endothelium by cytokines such as TNF and IL-1. Blocking LFA-1 on T cells with antibodies to either the a or ⁇ subunit strongly inhibits adhesion-dependent functions such as CTL-mediated lysis of target cells.
  • Mac-l (_ M 3 2 ) is distributed on neutrophils and monocytes, and its counter-receptor is also ICAM-1 (and possibly ICAM-2) .
  • Mac-l is the type 3 complement receptor (CR3) and binds the C3bi fragment.
  • the third 02 integrin, P150,95 (a ⁇ ⁇ 2 ) is also found on neutrophils and monocytes, but seems of less importance.
  • the ⁇ subunits of LFA-1, Mac-l and P150,95 are also given the respective CD designations CDlla, CDllb and CDllc, while ⁇ 2 is also denoted CD18, so that LFA-1 is CDlla/CD18 and Mac-l is CDllb/CD18.
  • selectins which were previously known as LECCAMs, and are now designated L- selectin (also called LECAM-1, Mel-14 or LAM-1) , E-selectin (also called ELAM-1) and P-selectin (also called GMP140 or PADGEM) .
  • L-selectin has a dual role: it is a homing receptor on T cells for the high endothelial venules of peripheral lymph nodes, and it is an adhesion molecule on neutrophils for endothelium (Hallmann et al., Biochem. Biophys. Res. Commun. 174, 236 (1991), which is incorporated herein by reference) .
  • E-selectin and P-selectin are both induced on endothelium by cytokines, although with different kinetics.
  • L-selectin is a counter-receptor on neutrophils for both E- selectin and P-selectin (Picker et al., Cell 66, 921 (1991), which is incorporated herein by reference) , although all three selectins probably have other counter-receptors as well.
  • E-selectin binds the carbohydrate group sialyl Lewis x (sLex) (Lowe et al., Cell 63, 475 (1990)), which is incorporated herein by reference) , and while this carbohydrate is prominently presented on L-selectin (Picker et al., Cell 66, 921 (1991)), it may occur on other proteins as well.
  • E-selectin is expressed especially in cutaneous sites of inflammation and also serves as an adhesion molecule for skin-homing T cells that may contribute to the inflammation (Picker et al. Nature 349, 796 (1991), which is incorporated herein by reference) .
  • antibodies to CDlla, CDllb, CD18, L-selectin and E-selectin all block binding of neutrophils to activated endothelial cells to a lessor or greater degree, but the most complete inhibition is generally achieved by the combination of an antibody to CD18 and an antibody to L- or E-selectin (see e.g., Luscinskas, J. Immunol. 142, 2257 (1989), which is incorporated herein by reference) .
  • Luscinskas J. Immunol. 142, 2257 (1989), which is incorporated herein by reference
  • a recent but now widely accepted model accounts for these facts with a three step process of adhesion (Butcher, Cell 67, 1033 (1991), which is incorporated herein by reference) .
  • neutrophils reversibly bind to inflamed vascular endothelium via the selectins, which bind well under conditions of flow, causing the neutrophils to literally roll along the vascular wall.
  • the neutrophils are then activated by a variety of stimulants surrounding or released by the endothelium, including IL-8, PAF and C5a.
  • the activated neutrophils shed L-selectin and up-regulate Mac-l.
  • binding of Mac-l to ICAM-1 and perhaps other counter-receptors on the endothelial cells allows stable adhesion and extravasation through the endothelium.
  • neutrophils Although important for eliminating infection, neutrophils are now believed to be a major cause of tissue damage during inflammation by binding to vascular endothelium and migrating through it into tissues (Harlan, Acta. Med. Scand. Suppl. 715, 123 (1987), which is incorporated herein by reference) .
  • the neutrophils release proteases and toxic, reactive oxygen metabolites which damage endothelium as well as other tissues.
  • antibodies or other antagonists of the integrin and selectin adhesion molecules could abort this process, by preventing neutrophils from binding to endothelium and from extravasating into tissues. Hence such antibodies could be used to treat a great many different disease conditions of which inflammation is an important component.
  • anti-CD18 antibodies which bind to both LFA-1 and Mac-l, have been especially useful in reducing ischemia-reperfusion injury (see, e.g., Vedder et al. , J. Clin. Invest. 81, 939 (1988); Vedder et al., Proc. Natl. Acad. Sci. USA 87, 2643 (1990); U.S. Patent No. 4,797,277). They also reduce neutrophil-mediated damage in the lung in response to various insults (Doerschuk et al., J. Immunol. 144, 2327 (1990) and Mulligan et al., J. Immunol.
  • anti-CD18 antibodies also protect from lethality due to meningitis (Tuomanen et al., J. Exp. Med. 170, 959 (1990)). They may also be useful in preventing or treating organ transplant rejection because they block T-cell function. For example, injection of antibodies to L-selectin or E-selectin into rodents suppressed neutrophil accumulation within inflamed peritoneum (Jutila et al. J. Immunol. 143, 3318 (1989) and Mulligan et al., J. Clin. Invest.
  • an anti-E-selectin antibody greatly reduced neutrophil influx into the lung and associated late-phase airway obstruction after antigen inhalation (Gundel et al., J. Clin. Invest. 88, 1407 (1991)).
  • the antibody NA-8 has been developed that binds to human CD18 as disclosed herein. This antibody partially or completely blocks the binding of human neutrophils to stimulated human umbilical vein endothelial cells. Because of its ability to block binding of neutrophils to endothelial cells, this antibody could be used to treat inflammatory disease conditions (see above) .
  • non-human monoclonal antibodies contain substantial stretches of amino acid sequences that will be immunogenic when injected into a human patient. Numerous studies have shown that, after injection of a foreign antibody, the immune response elicited by a patient against an antibody can be quite strong, essentially eliminating the antibody's therapeutic utility after an initial treatment. Moreover, as increasing numbers of different mouse or other antigenic (to humans) monoclonal antibodies can be expected to be developed to treat various diseases, after the first or several treatments with any different non-human antibodies, subsequent treatments even for unrelated therapies can be ineffective or even dangerous in themselves, because of cross-reactivity.
  • the present invention provides novel compositions useful, for example, in the treatment of inflammatory human disorders, the compositions containing mouse or humanized immunoglobulins specifically capable of binding to CD18.
  • the immunoglobulins can have two pairs of light chain/heavy chain complexes, at least one chain comprising one or more mouse complementarity determining regions functionally joined to human framework region segments.
  • mouse complementarity determining regions with or without additional naturally-associated mouse amino acid residues, can be introduced into human framework regions to produce humanized immunoglobulins capable of binding to the CD18 at affinity levels stronger than about 10 7 M -1 .
  • These humanized immunoglobulins will also be capable of blocking the binding of the CDR-donating mouse monoclonal antibody to CD18.
  • the immunoglobulins, including binding fragments and other derivatives thereof, of the present invention may be produced readily by a variety of recombinant DNA techniques, with ultimate expression in transfected cells, preferably immortalized eukaryotic cells, such as myeloma or hybridoma cells.
  • Polynucleotides comprising a first sequence coding for humanized immunoglobulin framework regions and a second sequence set coding for the desired immunoglobulin complementarity determining regions can be produced synthetically or by combining appropriate cDNA and genomic DNA segments.
  • the humanized immunoglobulins may be utilized alone in substantially pure form, or together with a chemotherapeutic agent such as a non-steroidal anti- inflammatory drug, a corticosteroid, or an immunosuppressant. All of these compounds will be particularly useful in treating inflammatory disorders.
  • a chemotherapeutic agent such as a non-steroidal anti- inflammatory drug, a corticosteroid, or an immunosuppressant. All of these compounds will be particularly useful in treating inflammatory disorders.
  • the humanized immunoglobulins or their complexes can be prepared in a pharmaceutically acceptable dosage form, which will vary depending on the mode of administration.
  • FIGURES Fig. 1 Sequences of the cDNA and translated amino acid sequences of the light chain (A) [SEQ. ID NOS:l and 2] and heavy chain (B) [SEQ. ID NOS:3 and 4] variable regions of the NA-8 antibody.
  • the mature light chain begins with amino acid 21 D, and the mature heavy chain begins with amino acid 20 Q, preceded by the respective signal sequences.
  • Fig. 2 Amino acid sequences of the mature light chain (A) [SEQ. ID NO:5] and heavy chain (B, [SEQ. ID NO:6] C [SEQ. ID NO:7]) variable regions of the mouse NA-8 antibody (upper lines) and humanized NA-8 antibody (B; lower lines) and humanized NA-8a antibody (C, lower lines) .
  • A mature light chain
  • B heavy chain
  • C C
  • SEQ. ID NO:7 C
  • CDRs in each chain are underlined. Residues in the framework that have been replaced with mouse amino acids or typical human amino acids in the humanized antibody are double underlined.
  • Fig. 3. Nucleotide sequences of the genes encoding the light chain (A) [SEQ. ID NOS:8 and 9] and heavy chain (B) [SEQ. ID NOS:10 and 11] variable regions of the humanized NA- 8 antibody, beginning and ending with the Xbal sites, and translated amino acid sequences, including signal sequences.
  • Fig. 4. Competitive binding of mouse and humanized
  • the target cells were THP-1 cells, a human monocytic line that expresses human CD18 (ATCC TIB 202). 2 x 10 5 cells were incubated with 3 ng of 1 5 I- labeled tracer mouse antibody (2 ⁇ Ci/ ⁇ g) , together with increasing amounts of mouse or humanized competitor antibody as indicated in 0.2 ml of binding buffer (PBS + 2% FBS + 0.1% azide) for 1 hr at 4°C. Cells were washed and pelleted, and their bound radioactivity measured. The concentrations of bound and free tracer antibody were calculated. Fig. 5. Binding of human neutrophils to IL-1 stimulated human umbilical cord endothelial cells (HUVEC) . The neutrophils were first treated with irrelevant control antibody, mouse NA-8 antibody, or humanized IgGl NA-8 antibody, as indicated.
  • VEC human umbilical cord endothelial cells
  • naturally-occurring refers to the fact that an object can be found in nature.
  • a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally-occurring.
  • substantially pure means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition) , and preferably a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition will comprise more than about 80 to 90 percent of all macromolecular species present in the composition. Most preferably, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.
  • label refers to incorporation of a detectable marker, e.g.. by incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods) .
  • marked avidin e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods
  • Various methods of labeling polypeptides and glycoproteins are known in the art and may be used.
  • labels for polypeptides include, but are not limited to, the following: radioisotopes (e.g., 3 H, 14 C, 32 P, 35 S, 125 I, 131 I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, ⁇ -galactosidase, luciferase, alkaline phosphatase) , biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags) .
  • labels are attached by spacer arms of various lengths to reduce potential steric hindrance.
  • mouse or humanized immunoglobulins specifically reactive with CD18 related epitopes are provided.
  • These immunoglobulins which have binding affinities to CD18 under suitable binding conditions (e.g., physiological serum conditions) of at least about 10 7 M _1 , and preferably 10 8 M "1 to 10 10 M _1 or stronger, are capable of, e.g.. binding to CDl8-expressing cells , such as neutrophils.
  • the humanized immunoglobulins will have a human framework and will have one or more complementarity determining regions (CDRs) from an immunoglobulin, typically a mouse immunoglobulin, specifically reactive with CD18.
  • CDRs complementarity determining regions
  • one or more of the CDRs will come from the NA-8 antibody, and the humanized immunoglobulin will be of the IgGl or IgG4 isotype.
  • the immunoglobulins of the present invention which can be produced economically in large quantities, find use, for example, in the treatment of adhesion-related pathological conditions (e.g., inflammatory disorders) in human patients by a variety of techniques.
  • the basic antibody structural unit is known to comprise a tetramer.
  • Each tetramer is composed of two iden ⁇ tical pairs of polypeptide chains, each pair having one "light” (about 25kD) and one "heavy" chain (about 50-70kD) .
  • the NH 2 -terminus of each chain begins a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the COOH part of each chain defines a constant region primarily responsible for effector function.
  • Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the antibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively.
  • the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D” region of about 10 more amino acids.
  • the variable regions of each light/heavy chain pair form the antibody binding site.
  • the chains all exhibit the same general structure of relatively conserved framework regions joined by three hypervariable regions, also called Complementarity Determining Regions or CDRs (see, "Sequences of Proteins of Immunological Interest,” Rabat, E. , et al. , U.S. Department of Health and Human Services, (1987); and Chothia and Lesk, J. Mol. Biol., 196. 901-917 (1987), which are incorporated herein by reference) .
  • the CDRs from the two chains of each pair are aligned by the framework regions, enabling binding to a specific epitope.
  • immunoglobulin refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes.
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes.
  • the immunoglobulins may exist in a variety of forms besides antibodies; including, for example, Fv, Fab, and (Fab') 2 as well as bifunctional hybrid antibodies (e.g., Lanzavecchia et al. , Eur. J. Immunol. 17 . 1° 5 (1987)) and in single chains (e.g.. Huston et al.
  • variable immunoglobulins will vary somewhat in terms of length by deletions, substitutions, insertions or additions of one or more amino acids in the sequences.
  • variable and constant regions are subject to substantial natural modification, yet are “substantially identical” and still capable of retaining their respective activities.
  • Human constant region and rearranged variable region DNA sequences can be isolated in accordance with well known procedures from a variety of human cells, but preferably immortalized B- cells. Similar methods can be used to isolate nonhuman immunoglobulin sequences from non-human sources.
  • Suitable source cells for the DNA sequences and host cells for expression and secretion can be obtained from a number of sources, such as the American Type Culture Collection ("Catalogue of Cell Lines and Hybrido as,” Fifth edition (1985) Rockville, Maryland, U.S.A., which is incorporated herein by reference) .
  • substantially identical modified heavy and light chains can be readily designed and manufactured utilizing various recombinant DNA techniques well known to those skilled in the art.
  • the chains can vary from the naturally-occurring sequence at the primary structure level by several amino acid substitutions, terminal and intermediate additions and deletions, and the like.
  • polypeptide fragments comprising only a portion of the primary structure may be produced, which fragments possess one or more immunoglobulin activities (e.g.. binding activity) .
  • the immunoglobulin-related genes contain separate functional regions, each having one or more distinct biological activities.
  • modifications of the genes encoding the desired epitope binding components may be readily accomplished by a variety of well-known techniques, such as site-directed mutagenesis (see. Gillman and Smith, Gene 8:81-97 (1979) and Roberts, S. et al., Nature 328:731- 734 (1987) , both of which are incorporated herein by reference) .
  • the epitope binding component is encoded by immunoglobulin genes that are "chimeric” or "humanized” (see, generally, Co and Queen (1991) Nature 351:501. which is incorporated herein by reference) .
  • the humanized antibodies of the invetion will comprise heavy chain variable region sequences wherein the sequence of the humanized immunoglobulin heavy chain variable region framework is more than 65% identical but less than 95% identical to the sequence of the donor immunoglobulin heavy chain variable region framework, preferably the variable region framework is less than 10 % identical to the sequence of the donor immunoglobulin.
  • Chimeric antibodies are antibodies whose light and heavy chain genes have been constructed, typically by genetic engineering, from immunoglobulin gene segments belonging to different species.
  • the variable (V) segments of the genes from a mouse monoclonal antibody may be joined to human constant (C) segments, such as ⁇ -_ and y 4 .
  • a typical therapeutic chimeric antibody is thus a hybrid protein consisting of the V or antigen-binding domain from a mouse antibody and the C or effector domain from a human antibody, although other mammalian species may be used.
  • framework region refers to those portions of immunoglobulin light and heavy chain variable regions that are relatively conserved (i.e.. other than the CDRs) among different immunoglobulins in a single species, as defined by Kabat, et al., OJD. cit.
  • a "human framework region” is a framework region that is substantially identical (about 85% or more) to the framework region of a naturally occurring human antibody.
  • humanized immunoglobulin refers to an immunoglobulin comprising a human framework, at least one CDR from a non-human antibody, and in which any constant region present is substantially identical to a human immunoglobulin constant region, i.e.. at least about 85-90%, preferably at least 95% identical.
  • all parts of a humanized immunoglobulin, except possibly the CDRs are substantially identical to corresponding parts of one or more native human immunoglobulin sequences.
  • a humanized immunoglobulin would not encompass a chimeric mouse variable region/human constant region antibody.
  • Humanized antibodies have at least three potential advantages over mouse and in some cases chimeric antibodies for use in human therapy:
  • the effector portion is human, it may interact better with the other parts of the human immune system (e.g.. destroy the target cells more efficiently by complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC) ) .
  • CDC complement-dependent cytotoxicity
  • ADCC antibody-dependent cellular cytotoxicity
  • the human immune system should not recognize the framework or C region of the humanized antibody as foreign, and therefore the antibody response against such an injected antibody should be less than against a totally foreign mouse antibody or a partially foreign chimeric antibody.
  • Injected mouse antibodies have been reported to have a half-life in the human circulation much shorter than the half-life of normal antibodies (Shaw, D. et al., J. Immunol. 138. 4534-4538
  • Injected humanized antibodies will presumably have a half-life more like that of naturally occurring human antibodies, allowing smaller and less frequent doses to be given.
  • the present invention is directed to recombinant DNA segments encoding the heavy and/or light chain CDRs from an immunoglobulin capable of binding to a desired epitope of CD18, such as monoclonal antibody NA-8.
  • the DNA segments encoding these regions will typically be joined to DNA segments encoding appropriate human framework regions.
  • Exemplary DNA sequences, which on expression code for the polypeptide chains comprising the heavy and light chain CDRs of monoclonal antibody NA-8 are included in Fig. i. Due to codon degeneracy and non-critical amino-acid substitutions, othar DNA sequences can be readily substituted for those sequences, as detailed below. For a detailed description of the design and production of humanized immunoglobulins, see, commonly assigned serial nos.
  • the DNA segments will typically further include an expression control DNA sequence operably linked to the humanized immunoglobulin coding sequences, including naturally-associated or heterologous promoter regions.
  • the expression control sequences will be eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells, but control sequences for prokaryotic hosts may also be used.
  • nucleic acid sequences of the present invention capable of ultimately expressing the desired humanized antibodies can be formed from a variety of different polynucleotides (genomic or cDNA, RNA, synthetic oligonucleotides, etc.) and components (e.g. , V, J, D, and C regions) , as well as by a variety of different techniques.
  • Human constant region DNA sequences can be isolated in accordance with well known procedures from a variety of human cells, but preferably immortalized B-cells (see. Kabat op., cit. and WP87/02671) .
  • the CDRs for producing the immunoglobulins of the present invention will be similarly derived from monoclonal antibodies capable of binding to CD18 and produced in any convenient mammalian source, including, mice, rats, rabbits, or other vertebrate capable of producing antibodies by well known methods.
  • Suitable source cells for the DNA sequences and host cells for immunoglobulin expression and secretion can be obtained from a number of sources, such as the American Type Culture Collection (Catalogue of Cell Lines and Hvbridomas.
  • the CDRs have sequences corresponding to the CDR sequences of NA-8, and may include degenerate nucleotide sequences encoding, the corresponding CDR amino acid sequence(s) of NA-8.
  • the framework regions can vary from the native sequences at the primary structure level by several amino acid substitutions, terminal and intermediate additions and deletions, and the like.
  • a variety of different human framework regions may be used singly or in combination as a basis for the humanized immunoglobulins of the present invention.
  • modifications of the genes may be readily accomplished by a variety of well-known techniques, such as site-directed mutagenesis (see. Gillman and Smith, Gene 8.,
  • polypeptide fragments comprising only a portion of the primary antibody structure may be produced, which fragments possess one or more immunoglobulin activities (e.g.. binding activity) .
  • These polypeptide fragments may be produced by proteolytic cleavage of intact antibodies by methods well known in the art, or by inserting stop codons at the desired locations in the vectors pVk and pVgl-dhfr using site-directed mutagenesis, such as after CHI to produce Fab fragments or after the hinge region to produce (Fab*) 2 fragments.
  • Single chain antibodies may be produced by joining VL and VH with a DNA linker (see Huston et al., op cit.. and Bird et al., op_cit.).
  • Fv or Fab fragments may be produced in E. coli according to the methods of Buchner and Rudolph (1991) Bio/Technology 9.: 157-162 and Skerra et al. (1991) Bio/Technology : 273-277, incorporated herein by reference) .
  • Fv and Fab may also be produced by expression of endoding polynucleotides in eukaryotic, preferably mammalian, cells.
  • the immunoglobulin-related genes contain separate functional regions, each having one or more distinct biological activities, the genes may be fused to functional regions from other genes (e.g.. enzymes, see, commonly assigned U.S.S.N. 132,387, filed Dec.
  • fusion proteins e.g.. immunotoxins
  • Expression of the humanized immunoglobulin sequences in bacterial hosts may be used to advantage to select higher affinity humanized immunoglobulin sequences by mutagenizing the CDR regions and producing bacteriophage display libraries which may be screened for humanized immunoglobulin CDR variants which possess high affinity and/or high specificity binding to CD18.
  • affinity sharpening is the generation of humanized immunoglobulin CDR variants which have improved binding affinity and/or reduced cross-reactivity with molecules other than CD18s.
  • the DNA sequences will be expressed in hosts after the sequences have been operably linked to (i.e.. positioned to ensure the functioning of) an expression control sequence.
  • These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA.
  • expression vectors will contain selection markers, e.g.. tetracycline-resistance (tet R ) , G418-resistance (neo R ) , ycophenolic acid-resistance (gpt) , or HSV-tk, to permit detection of those cells transformed with the desired DNA sequences (see, e.g. , U.S. Patent 4,704,362, which is incorporated herein by reference) .
  • E. coli is one prokaryotic host useful particularly for cloning the DNA sequences of the present invention.
  • Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis. and other enterobacteriaceae, such as Salmonella. Serratia. and various Pseudomonas species.
  • bacilli such as Bacillus subtilis.
  • enterobacteriaceae such as Salmonella. Serratia. and various Pseudomonas species.
  • expression vectors which will typically contain expression control sequences compatible with the host cell (e.g. , an origin of replication) .
  • any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda.
  • the promoters will typically control expression, optionally with an operator sequence, and have ribosome binding site sequences and the like, for initiating and completing transcription and translation.
  • Saccharomvces is a preferred host, with suitable vectors having expression control sequences, such as promoters, including 3-phosphoglycerate kinase or other glycolytic enzymes, and an origin of replication, termination sequences and the like as desired.
  • Plants and plant cell cultures may be used for expression of the humanized immunoglobulins of the invention.
  • Preferable plant hosts include, for example: Arabidopsis. Nicotiana tabacum, Nicotiana rustica. and Solanum tuberosum.
  • a preferred expression cassette for expressing polynucleotide sequences encoding the humanized anti-CD18 antibodies of the invention is the plasmid pMOG18 in which the inserted polynucleotide sequence encoding the humanized immunoglobulin chain is operably linked to a CaMV 35S promoter with a duplicated enhancer; pMOG18 is used according to the method of Sijmons et al. (1990) Bio/Technology 8 . : 217-221, incorporated herein by reference.
  • pMOG18 is used according to the method of Sijmons et al. (1990) Bio/Technology 8 . : 217-221, incorporated herein by reference.
  • a preferred embodiment for the expression of humanized immunoglobulins in plants follows the methods of Hiatt et al. (1989) op.cit..
  • Agrobacterium tumifaciens T-DNA-based vectors may also be used for expressing humanized immunoglobulin sequences, preferably such vectors include a marker gene encoding spectinomycin-resistance or other selectable marker.
  • Insect cell culture may also be used to produce the humanized immunoglobulins of the invention, typically using a baculovirus-based expression system.
  • the humanized immunoglobulins may be produced by expressing polynucleotide sequences encoding the humanized immunoglobulins according to the methods of Putlitz et al. (1990) Bio/Technology 8 : 651- 654, incorporated herein by reference. The method of Putlitz et al. can be followed with the modification that polynucleotide sequences encoding the humanized anti-CD18 antibodies of the invention are inserted in place of the mouse monoclonal Ab 6A4 heavy chain and light chain cDNA sequences of Putlitz et al.
  • mammalian tissue cell culture may also be used to express and produce the polypeptides of the present invention (see. Winnacker, From Genes to Clones. VCH Publishers, N.Y., N.Y. (1987), which is incorporated herein by reference) .
  • Mammalian cells are actually preferred, because a number of suitable host cell lines capable of secreting intact immunoglobulins have been developed in the art, and include the CHO cell lines, various COS cell lines, HeLa cells, preferably myeloma cell lines, etc, or transformed B-cells or hybridomas.
  • Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer (Queen et al., Immunol. Rev. 8 _, 49-68 (1986) , which is incorporated herein by reference) , and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences.
  • Preferred expression control sequences are promoters derived from immunoglobulin genes, SV40, Adenovirus, Bovine Papilloma Virus, cytomegalovirus and the like.
  • a selectable marker such as a neo R expression cassette, is included in the expression vector.
  • Transgenes encoding a humanized immunoglobulin of the invention may be used to generate transgenic nonhuman animals which express the desired humanized immunoglobulin, typically in a recoverable body fluid such as milk or serum.
  • Such transgenes comprise a polynucleotide sequence encoding the humanized immunoglobulin(s) operably linked to a promoter, usually with a linked enhancer, such as a rodent immunoglobulin enhancer or a casein gene promoter/enhancer (Buhler et al. (1990) Bio/Technology 8 . : 140-143; Meade et al. (1990) Bio/Technology 8.: 443-446, incorporated herein by reference) .
  • Transgenes may be transferred into cells and embryos according to the methods described in the art and infra for homologous recombination constructs.
  • Preferred nonhuman animals include: mice, rats, sheep, cows, and goats; with expression in bovine milk being particularly preferred.
  • Purification of the humanized antibodies is accomplished by art-known purification methods for immunoglobulin purification.
  • Homologous recombination constructs may also be used for producing cells or transgenic animals expressing the humanized immunoglobulins of the invention.
  • Preferred promoter/enhancer combinations for operable linkage to polynucleotide sequences encoding the humanized immunoglobulins of the invention in the homologous recombination constructs include those associated with the albumin gene, 3-casein gene, ⁇ -casein gene, whey protein genes, or lactalbumin gene.
  • a selectable marker gene expression cassette e.g., neo R , HSV-tk, or gpt operably linked to a constitutive promoter such as the pgk gene promoter
  • the homologous targeting constructs encoding humanized immunoglobulins of the invention can be transferred into embryonic stem (ES) cells by lipofection, electroporation, or needle microinjection, or into fertilized animal embryos by pronuclear needle microinjection, or other art-accepted method.
  • ES cells are used for transgenesis, the resultant ES cells are usually selected by positive selection, and optionally be negative selection as well, and verified for correctly targeted recombination by PCR or Southern blotting. Correctly targeted ES cells are incorporated into blastocysts by blastocyst injection according to methods known in the art and cited herein. General principles regarding the construction of targeting constructs and selection methods are reviewed in Bradley et al. (1992) Bio/Technology 10: 534, incorporated herein by reference.
  • Chimeric targeted mice are derived according to Hogan, et al. , Manipulating the Mouse Embrvo: A Laboratory Manual. Cold Spring Harbor Laboratory (1988) and Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E.J. Robertson, ed. , IRL Press, Washington, D.C., (1987) which are incorporated herein by reference.
  • Embryonic stem cells are manipulated according to published procedures (Teratocarcinomas and Embryonic Stem Cells: A Practical Approach , E.J. Robertson, ed. , IRL Press, Washington, D.C. (1987); Zjilstra et al., Nature 342:435-438 (1989); and Schwartzberg et al.. Science 246:799-803 (1989), each of which is incorporated herein by reference) .
  • homologous targeting constructs are propagated in cloning hosts (e.g. , bacteria, yeast) and may also include an origin of replication and selectable marker(s) for facile cloning. It is frequently preferable to use a transfection technique with linearized transgenes containing only modified target gene sequence(s) and without vector sequences.
  • the modified gene site is such that a homologous recombinant between the exogenous targeting construct and the endogenous DNA target sequence can be identified by using carefully chosen primers and PCR or by Southern blot analysis, followed by analysis to detect if PCR products or Southern blot bands specific to the desired targeted event are present (Erlich et al., (1991) Science 252: 1643, which is incorporated herein by reference) .
  • ES cells embryonal stem cells
  • Murine ES cells such as AB-l line grown on mitotically inactive SNL76/7 cell feeder layers (McMahon and Bradley, Cell 62:1073-1085 (1990)) essentially as described (Robertson, E.J. (1987) in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. E.J. Robertson, ed. (Oxford: IRL Press) , p. 71-112) may be used for homologous gene targeting.
  • Other suitable ES lines include, but are not limited to, the E14 line (Hooper et al.
  • the blastocysts containing the injected ES cells are allowed to develop in the uteri of pseudopregnant nonhuman females and are born as chimeric mice.
  • the resultant transgenic mice are chimeric for cells having the homologously integrated humanized immunoglobulin gene sequence(s) and are backcrossed and screened for the presence of the correctly targeted transgene(s) by PCR or Southern blot analysis on tail biopsy DNA of offspring so as to identify transgenic mice heterozygous for the inactivated lymphocyte transduction locus/loci.
  • the vectors containing the DNA segments of interest can be transferred into the host cell by well-known methods, which vary dependinq on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment, lipofection, biolistics, viral-based transduction, or electroporation may be used for other cellular hosts. Tungsten particle ballistic transgenesis is preferred for plant cells and tissues. (See. generally. Maniatis et al. , Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press, (1982) , which is incorporated herein by reference.)
  • the whole antibodies, their dimers, individual light and heavy chains, or other immunoglobulin forms of the present invention can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like (see, generally. Scopes, R. , Protein Purification. Springer-Verlag, N.Y. (1982), which is incorporated herein by reference) .
  • Substantially pure immunoglobulins of at least about 90 to 95% homogeneity are preferred, and 98 to 99% or more homogeneity most preferred, for pharmaceutical uses.
  • polypeptides may then be used therapeutically (including extracorporeally) or in developing and performing assay procedures, immunofluorescent stainings, and the like. (See, generally. Immunological Methods. Vols. I and II, Lefkovits and Pernis, eds. , Academic Press, New York, N.Y. (1979 and 1981)).
  • humanized immunoglobulins are produced which bind to CD18 with a binding affinity of at least 1 x 10 7 M _1 in standard binding conditions (e.g., phosphate-buffered saline with 2 percent fetal bovine serum at 25°C) ; one example of such humanized immunoglobulins is the humanized NA-8 antibody comprising the amino acid sequences shown in Fig. 2.
  • the humanized antibodies of the invention preferably bind, in standard binding conditions, to human CD18 with an affinity of at least 1 x 10 8 M "1 , more preferably with an affinity of at least 1 x 10 9 M -1 , and advantageously with an affinity of at least 1 x 10 10 M "1 or stronger.
  • the antibodies of the present invention will typically find use in the treatment of disease conditions with an inflammatory component, especially those which are mediated by neutrophils or T cells.
  • a preferred application is the treatment of ischemia-reperfusion injury caused by myocardial infarction, cerebral ischemic event (e.g., stroke) , renal infarction, brain surgery, shock, cardiac surgery (e.g., coronary artery bypass), elective angioplasty, and the like.
  • Other preferred applications are the treatment of sepsis, adult respiratory distress syndrome, and multiple organ failure.
  • the antibodies will find use in treating injury due to trauma, burns, frostbite or damage to the spinal cord.
  • autoimmune diseases including by way of example and not limitation, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, type I diabetes and uveitis, in treating inflammatory diseases of the skin such as psoriasis, and in treating meningitis and encephalitis.
  • Other typical applications are the prevention and treatment of organ transplant rejection, graft-versus-host disease, and neoplasia.
  • any humanized immunoglobulins of the present invention may also be used in combination with other antibodies, particularly humanized antibodies reactive with different adhesion molecules.
  • suitable antigens to which a cocktail of humanized immunoglobulins may react include CDlla, CDllb, L-selectin, E-selectin, P-selectin and ICAM-1.
  • suitable antigens are ly phokines such as IL-1, IL-2 and IFN-7, and their receptors.
  • the humanized antibodies can also be used as separately administered compositions given in conjunction with chemotherapeutic agents.
  • the agents may include non-steroidal anti-inflammatory drugs and corticosteroids, but numerous additional agents (e.g., cyclosporin) well-known to those skilled in the art of medicine may also be utilized.
  • the humanized immunoglobins of the present invention will typically be used in combination with drugs currently used by those skilled in the art to treat particular diseases.
  • a preferred pharmaceutical composition of the present invention comprises the use of the subject immunoglobulins in immunotoxins to kill CD18 expressing cells.
  • Immunotoxins are characterized by two components and are particularly useful for killing selected cells in vitro or ill vivo.
  • One component is a cytotoxic agent which is usually fatal to a cell when attached or absorbed.
  • the second component known as the "delivery vehicle,” provides a means for delivering the toxic agent to a particular cell type, such as cells expressing a CD18 epitope.
  • the two components are commonly chemically bonded together by any of a variety of well-known chemical procedures.
  • the linkage may be by way of heterobifunctional cross-linkers, e.g.. SPDP, carbodiimide, glutaraldehyde, or the like.
  • heterobifunctional cross-linkers e.g.. SPDP, carbodiimide, glutaraldehyde, or the like.
  • Production of various immunotoxins is well-known with the art, and can be found, for example in "Monoclonal Antibody-Toxin Conjugates: Aiming the Magic Bullet," Thorpe et al. , Monoclonal Antibodies in Clinical Medicine. Academic Press, pp. 168-190 (1982) , which is incorporated herein by reference.
  • the components may also be linked genetically (see Chaudhary et al. , Nature 339, 394 (1989) , incorporated herein by reference) .
  • Cytotoxic agents are suitable for use in immunotoxins.
  • Cytotoxic agents can include radionuclides, such as Iodine-131 or other isotopes of iodine, Yttrium-90, Rhenium-188, and Bismuth-212 or other alpha emitters; a number of chemotherapeutic drugs, such as vindesine, methotrexate, adriamycin, and cisplatin; and cytotoxic proteins such as ribosomal inhibiting proteins like pokeweed antiviral protein, Pseudomonas exotoxin A, ricin, diphtheria toxin, ricin A chain, etc., or an agent active at the cell surface, such as the phospholipase enzymes (e.g..).
  • radionuclides such as Iodine-131 or other isotopes of iodine, Yttrium-90, Rhenium-188, and Bismuth-212 or other alpha emitters
  • the delivery component of the immunotoxin will include the humanized immunoglobulins of the present invention. Intact immunoglobulins or their binding fragments, such as Fab or Fv, are preferably used. Typically, the antibodies in the immunotoxins will be of the human IgM or IgG isotype, but other mammalian constant regions may be utilized as desired.
  • the humanized anti-CD-18 antibodies of the present invention will comprise a detectable label; typically the label is attached by covalent linkage or incorporation of a labeled radionuclide into the glycoprotein immunoglobulin.
  • a labeled second antibody e.g., goat anti-human IgG
  • This approach can be used advantageously when the sample being assayed for CD-18 expressing cells also contains other species which would interfere with a mouse primary antibody reactive with CD-18.
  • the humanized antibodies and pharmaceutical compositions thereof of this invention are particularly useful for parenteral administration, i.e.. subcutaneously, intramuscularly or intravenously.
  • the humanized antibodies of the invention may also be administered, typically for local application, by gavage or lavage, intraperitoneal injection, opthalmic ointment, topical ointment, intracranial injection (typically into a brain ventricle) , intrapericardial injection, or intrabursal injection.
  • the compositions for parenteral administration will commonly comprise a solution of the immunoglobulin or a cocktail thereof dissolved in an acceptable carrier, preferably an aqueous carrier.
  • an aqueous carrier preferably an aqueous carrier.
  • a variety of aqueous carriers can be used, e.g..
  • compositions may be sterilized by conventional, well-known sterilization techniques.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc.
  • concentration of antibody in these formulations can vary widely, i.e.. from less than about 0.005%, usually at least about 1% to as much as 15 or 20% by weight and will be selected primarily based on fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.
  • a typical pharmaceutical composition for injection could be made up to contain 1 ml sterile buffered water, and 1-10 mg of immunoglobulin.
  • a typical composition for intravenous infusion could be made up to contain 250 ml of sterile Ringer's solution, and 150 mg of antibody.
  • Actual methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art and are described in more detail in, for example, Remington's Pharmaceutical Science, 15th ed. , Mack Publishing Company, Easton, Pennsylvania (1980) , which is incorporated herein by reference. Compositions suitable for lavage or other routes will be selected according to the particular use intended.
  • the antibodies of this invention can be frozen or lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional immune globulins and art-known lyophilization and reconstitution techniques can be employed. It will be appreciated by those skilled in the art that lyophilization and reconstitution can lead to varying degrees of antibody activity loss (e.g.. with conventional immune globulins, IgM antibodies tend to have greater activity loss than IgG antibodies) and that use levels may have to be ad ⁇ justed to compensate.
  • compositions containing the present humanized antibodies or a cocktail thereof can be administered for prophylactic and/or therapeutic treatments.
  • compositions are administered to a patient already suffering from an inflammatory disease, in an amount sufficient to cure or at least partially arrest the disease and its complications.
  • An amount adequate to accomplish this is defined as a "therapeutically effective dose.” Amounts effective for this use will depend upon the severity of the disease and the general state of the patient's own immune system, but generally range from about 1 to about 200 mg of antibody per dose, with dosages of from 5 to 100 mg per patient being more commonly used.
  • Dosing schedules will vary with the disease state and status of the patient, and will typically range from a single bolus dosage or continuous infusion to multiple administrations per day (e.g., every 4-6 hours) , or as indicated by the treating physician and the patient's condition. It must be kept in mind that the mate ⁇ rials of this invention may generally be employed in serious disease states, that is life-threatening or potentially life- threatening situations. In such cases, in view of the minimization of extraneous substances and the lower probability of "foreign substance" rejections which are achieved by the present humanized immunoglobulins of this in ⁇ vention, it is possible and may be felt desirable by the treating physician to administer substantial excesses of these antibodies.
  • compositions containing the present antibodies or a cocktail thereof are administered to a patient not already suffering from a particular disease to enhance the patient's resistance.
  • Such an amount is defined to be a "prophylactically effective dose.”
  • the precise amounts again depend upon the patient's state of health and general level of immunity, but generally range from 1 to 50 mg per dose.
  • Preferred prophylactic uses are for the prevention of adult respiratory distress syndrome in patients already suffering from sepsis or trauma; prevention of organ transplant rejection; and prevention of reperfusion injury in patients suffering from ischemia. In seriously ill patients, dosages of about 50 to 100 mg of humanized immunoglobulin per administration are frequently used, and larger dosages may be indicated.
  • compositions can be carried out with dose levels and pattern being selected by the treating physician.
  • pharmaceutical formulations should provide a quantity of the antibody(ies) of this invention sufficient to effectively treat the patient.
  • Humanized antibodies of the present invention can further find a wide variety of utilities m vitro.
  • the antibodies can be utilized for detection of CD18 antigens, for isolating specific leukocytes, or the like.
  • a humanized NA-8 immunoglobulin can be immobilized and contacted with blood extravasated from a patient to remove blood cells bearing CD18 antigens, and the remaining blood, depleted of CD18- bearing cells, may be reintroduced into the patient. Any residual humanized antibody present in the depleted blood reintroduced into the patient (e.g., as a consequence of detachment from the immobilization support) would have reduced or negligible antigenicity as compared to a murine antibody.
  • the antibodies may either be labeled or unlabeled.
  • Unlabeled antibodies can be used in combination with other labeled antibodies (second antibodies) that are reactive with the humanized antibody, such as anti ⁇ bodies specific for human immunoglobulin constant regions.
  • second antibodies labeled antibodies
  • the antibodies can be directly labeled.
  • labels may be employed, such as radionuclides, fluors, enzymes, enzyme substrates, enzyme co- factors, enzyme inhibitors, ligands (particularly haptens) , etc.
  • Numerous types of immunoassays are available and are well known to those skilled in the art. The following examples are offered by way of illustration, not by limitation. It will be understood that although the examples pertain to the NA-8 antibody, producing humanized antibodies with high binding affinity for CD18 may also be performed using CDRs from other monoclonal antibodies that bind to an epitope of CD18.
  • the mouse antibody NA-8 that binds to human CD18 was developed as follows. A mouse was subcutaneously immunized with 25 x 10 6 PMA-activated human neutrophils emulsified in Hunter's TiterMaxTM adjuvant at two sites. The mouse was boosted 4 times by intraperitoneal injection of 20- 25 x 10 6 PMA-activated neutrophils at intervals of 2 to 3 weeks. Four days following the final boost, the spleen cells were fused with myeloma cell to produce hybridomas, following methods well known in the art. Supernatants of hybridomas were then screened for the ability to inhibit neutrophil adherence to protein-coated plastic. Hybridoma supernatant samples (100 ⁇ l) were transferred to a 96 well plate.
  • a neutrophil suspension (100 ⁇ l of 5 x 10 6 cells/ml) was added to each well. Following a 5 min incubation at room temperature, 20 ⁇ l of a 500 ng/ml stock of PMA was added to each well. The plate was incubated for thirty minutes at 37°C to allow the neutrophils to settle to the bottom of the well. Following this incubation, the wells of the plate were washed 3 times with RPMI to remove non-adherent cells. Each well was then stained with 50 ⁇ l of 0.2% crystal violet in
  • NA-8 specifically binds to CD18 was shown in two ways. Immunoprecipitation of labelled neutrophil lysate gave the same pattern as the Known anti-CD18 antibody IB4 (Wright et al. (1983) Proc. Natl. Acad. Sci. USA 80: 5699), that is bands of the correct size for CD18 and for co- precipitated CDllb and CDllc. Therefore, the antibody reacts with the CD11/CD18 complex, and the presence of 2 alpha chains (both CDllb and CDllc) implies that reactivity is in fact with CD18.
  • NA-8 binds to the same cell lines among a panel of cell lines as IB4. In particular, NA-8 reacts with Hut-102B and JURKAT cells as well as normal human peripheral blood lymphocytes, which express CD18 but not CDllb.
  • cDNAs for the heavy chain and light chain variable domain genes of NA-8 were cloned using anchored polymerase chain reactions as described (see Co .et al. , J. Immunol. 148, 1149 (1992) and commonly assigned U.S.S.N. 07/634,278), using 3 ' primers that hybridized to the constant regions and contained Hindlll sites, and 5' primers that hybridized to the dG tails and contained EcoRI sites. The PCR amplified fragments were digested with EcoRI and Hindlll and cloned into the pUC18 vector for sequencing.
  • NA-8 For NA-8, at least two gamma-1 specific and two kappa specific clones were sequenced. The gamma-1 clones and the kappa clones are respectively identical in sequence. The cDNA variable domain sequences and the deduced amino acid sequences are shown in
  • the computer program ENCAD (M. Levitt, J. Mol. Biol. 168. 595 (1983), which is incorporated herein by reference) was used to construct a model of the NA-8 variable region.
  • the model was used to determine the amino acids in the NA-8 framework that were close enough to the CDRs to potentially interact with them (category 4 below) .
  • the amino acids in each category are shown in Table 1. Some amino acids may be in more than one category.
  • the final sequences of the humanized NA-8 light and heavy chain variable domains are shown in Fig. 2, A-B compared with the murine NA-8 sequences.
  • amino acid from the mouse NA-8 antibody was chosen at light chain position 49, because it was at the light-heavy chain interface.
  • humanized NA-8a a second humanized version of NA-8 was constructed, designated humanized NA-8a.
  • This antibody has the same light chain as the first version (Fig. 2A) , but in the heavy chain the human EU amino acids were retained at positions 67, 68, 70 and 74, rather than being replaced by the murine NA-8 amino acids.
  • the sequence of the mature heavy chain variable region of humanized NA-8a is shown in Fig. 2C. Humanized NA-8a was produced and characterized analogously to humanized NA-8 (see below) .
  • nucleotide sequences were selected that encode the protein sequences of the humanized heavy and light chains, including signal peptides, generally utilizing codons found in the mouse sequence. Several degenerate codons were changed to create restriction sites or to remove undesirable ones.
  • the nucleotide sequences of the genes also included splice donor signals and an Xbal site at each end. For example, the nucleotide sequences and encoded humanized light and heavy chain variable domains of humanized NA-8 are shown in Fig. 3. Each gene was constructed from four overlapping synthetic oligonucleotides, as described (see. Co et al., J. Immunol.
  • the heavy and light chain variable region genes were then respectively ligated into the Xbal sites of the pVgl-dhfr or pVk expression vectors (see, commonly assigned U.S.S.N 07/634,278) in the appropriate orientations to produce the complete heavy and light chain genes. Reactions were carried out under conditions well-known in the art (Maniatis et al., op. cit.)
  • the heavy chain and light chain plasmids were transfected into Sp2/0 mouse myeloma cells by electroporation and cells were selected for gpt expression. Clones were screened by assaying human antibody production in the culture supernatant by ELISA, and antibody was purified from the best- producing clones. Humanized NA-8 IgGl antibody was then purified by passing tissue culture supernatant over a column of staphylococcal protein A-Sepharose CL-4B (Pharmacia) . The bound antibody was eluted with 0.2 M Glycine-HCl, pH3.0 and neutralized with 1 M Tris PH8.0. The buffer was exchanged into PBS by passing over a PD10 column (Pharmacia) , or by dialysis. To obtain cells producing higher levels of antibody, the transfected clones may be cultured in increasing concentrations of methotrexate.
  • the humanized NA-8 heavy chain variable region gene was then cloned into the Xbal site of pVg4-dhfr. This heavy chain plasmid was then transfected together with the above light chain plasmid into Sp2/0 cells, clones selected, and humanized NA-8 IgG4 antioody purified as described above for the IgGl antibody. A humanized NA-8a IgG4 antibody is produced similarly.
  • the affinity of the humanized NA-8 antibodies for CD18 were determined by competition with the radio-iodinated mouse NA-8 antibody (Fig. 4) .
  • the binding affinities were calculated according to the methods of Berzofsky (J.A.
  • the humanized NA-8 antibodies had an affinity within about 2-fold of the mouse NA-8 antibody. A similar result will be found when the affinity for CD18 on human neutrophils is measured.
  • the humanized NA-8a antibody was similarly determined to have approximately the same binding affinity for CD18 as humanized NA-8. The ability of the mouse and humanized NA-8 antibodies to block the adhesion of human neutrophils to endothelial cells was shown as follows (Fig 5) .
  • Human umbilical cord endothelial cells (HUVEC; from Clonetics, San Diego) were grown to confluency in EGM media (Clonetics) in a 24 well plate. Four hours prior to the assay, each well was washed with RPMI, and media containing 20 ng/ml IL-1/3 was added. The plates were then incubated at 37° C. Human neutrophils were isolated from buffy coats that had been cleared of erythrocytes by dextran sedimentation, and then adjusted to 10 7 cells per ml. The neutrophils were then labelled with 200-400 ⁇ Ci of 51 Cr.
  • the humanized immunoglobulins of the present invention offer numerous advantages over other CD18 specific antibodies.
  • the present humanized immunoglobulins can be more economically produced and contain substantially less foreign amino acid sequences.

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Abstract

On a préparé des immunoglobulines humanisées, réagissant spécifiquement avec la CD-18, par génie génétique, lesquelles sont utilisées pour traiter, notamment, des troubles inflammatoires.
PCT/US1993/011611 1992-12-01 1993-11-30 Anticorps humanises reagissant avec la cd-18 WO1994012214A1 (fr)

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